The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
DNA vaccination is a technique whereby somatic cells are transfected in vivo with DNA directing synthesis of a target antigen. Ulmer et al. disclose hereologous protection against influenza by injection of DNA encoding a viral protein (Science 259:1745, 1993). Watanabe et al. disclose the induction of antibodies to a kappa variable region by gene immunization (J. Immunol. 151:2871, 1993). The expressed protein either can be secreted by the transfected cell or processed inside the cell and presented in the context of class I major histocompatibility (MHC) antigens, which can be recognized by T cells. One of the pathways whereby polypeptides are processed into peptides involves intracellular proteolysis of the polypeptide into peptide fragments that ultimately bind MHC molecules. One major candidate process for this pathway is that of polyubiquitination.
Ubiquitination ("Ub"), an ATP-dependent process, constitutes a preliminary step of targeting a proteolytic substrate for its eventual degradation by the proteosome, a large multi-catalytic protease. Experiments in yeast and rabbit reticulocyte lysates indicate that at least two distinct determinants can dictate the rate of its degradation: one is the identity of N-terminal residue (N-end rule) and the other is presence of specific internal lysine residue where polyubiquitin is initiated (Bachmair, A., et al. Science 234:179-186, 1986; Gonda, D. K., et al. J. Biol. Chem. 264:16700-16712, 1989; Bachmair, A., et al. Cell 56:1019-1032, 1989). N-terminal amino acids are largely classified into three different categories based upon their destabilizing potential and the half-life of a given protein varies significantly (from 2 min to &gt;20h) depending on the identity of N-terminal amino acid (Bachmair, A., et al., Science 234:179-186, 1986; Gonda, D. K., et al. J. Biol. Chem. 264:16700-16712, 1989). Studies have indicated that intracellular degradation of proteins is required for antigen presentation to T cells (Townsend, A., et al. J. Exp. Med. 168:1211-1224, 1988; Townsend, A., et al. Cell 42:457-67, 1985; Germain, R. N. Cell 76:287-299, 1994).
Evidence that the Ub-mediated proteolytic pathway provides all of the substrates for the proteosome has remained inconclusive from temperature-sensitive UBEL mutant cells (Michalek, M. T., et al. Nature 363:552-554, 1993; Cox, J. H., et al. J. Immunol. 154:511-519, 1995). A recent study using pairs of N-end rule substrate proteins that varied in their intracellular stability provided evidence that the proteolytic turnover of endogenously synthesized proteins is not directly proportional to the generation of processed antigenic peptide/MHC class I complexes (Goth, S., et al. J. Immunol. 157:1894-1904, 1996). Goth et al., used the sindbis virus polymerase as the N-terminal target of the Ub-dependent degradation pathway. Earlier studies had shown that sindbis virus polymerase is a natural substrate for the N-rule (de Groot et al. Proc. Natl. Acad. Sci. USA, 88:8967, 1991).
Levy et al. (Proc. Natl. Acad. Sci. USA 93:4907, 1996) disclose a fusion protein consisting of a 21-kDa mouse DHFR moiety, an ubiquitin protein, a variable residue, 165 residues of nsP4 (Sinbis virus RNA polymerase) and .beta. gal that is useful in a method to produce equimolar amounts of two or more specific proteins in a cell.
Grant, E. P., et al. (J. Immunol. 155:3750-3758, 1995) disclose that chimeric proteins comprising ubiquitin, a destabilizing amino acid, a lacI extension and .beta. gal when loaded into LB27.4 cells (a B lymphoblastoid cell line) showed enhanced class I presentation compared to that of proteins with a stabilizing amino acid.
Bachmair et al. U.S. Pat. No. 5,496,721 disclose the use of genetic constructs that encode for ubiquitin fusion proteins with destabilizing amino acids at their N-termini.
A recent study found that ovalbumin (OVA) with methylated lysine groups which was resistant to ubiquitin-mediated degradation could still be presented via class I MHC, albeit at a reduced amount (Michalek, M. T., et al. J. Immunol. 157:617-624, 1996). This indicates that there may be a ubiquitin-independent pathway for class I presentation of antigens to the immune system.
WO 94/17816 disclose methods for the use of inhibitors of the ubiquitin-dependent proteolytic pathway to reduce cytolytic immune responses.